298 >

research

Factors affecting the preparation,

constituents, and clinical efficacy of
leukocyte- and platelet- rich fibrin
(L-PRF).
SADJ August 2016, Vol 71 no 7 p298 - p302

MT Peck1, D Hiss2, L Stephen3

Abstract
Platelet-rich fibrin (PRF) was first introduced by
Choukroun et al, in 2001 as a method of concentrating
autologous human leukocytes, platelets and fibrin
for autotransplantation into surgical wound sites to
accelerate healing. Even though several clinical reports
have documented the use of L-PRF, controversy still exists
with regards to many aspects of this biomaterial. Diverse
publications report the use of non-standardised methods
to prepare L-PRF, resulting in variable clinical results. The
impact of the type of centrifuge, as well as of the growth
factor release kinetics, have recently been studied and
have yielded new insights into the structure and function
of L-PRF. The presence of bone morphogenetic proteins
as well as stem cells has also been documented. In
this report we analyse various factors affecting L-PRF
preparation and its constituents and highlight some of the
controversies surrounding the biomaterial.
Introduction
Platelet-rich fibrin (PRF) was first introduced by
Choukroun et al. in 2001 as a method of concentrating
autologous human leukocytes, platelets and fibrin for
autotransplantation into surgical wound sites to accelerate
healing.1 This method of concentrating blood platelets was
different to previous techniques in that it centrifuged the
collected blood only once, no anticoagulant agents were
added, and leukocytes and fibrin were deliberately included
in the final product. Previous similar techniques had sought
1. Mogammad Thabit Peck: Department of Oral Medicine and
Periodontology, University of the Western Cape.
2. Donavon Hiss: Department of Medical Biosciences, University of
the Western Cape.
3. Lawrence Stephen: Department of Oral Medicine and
Periodontology, University of the Western Cape.
Corresponding author
Mogammad Thabit Peck:
Department of Oral Medicine and Periodontology, Faculty of Dentistry,
University of the Western Cape, Private Bag X1 Tygerberg, 7505,
South Africa. Tel: 021 9373186. Fax: 086 618 7560.
E-mail: mpeck@uwc.ac.za
ACRONYMs
BMPs: bone morphogenic proteins
G-CSF: granulocyte colony-stimulating factor
HUVEC: human umbilical vein endothelial cells
IGF-1: insulin-like growth factor-1
MSC: mesenchymal stem cells
PRF: platelet-rich fibrin
PDGF-AB: platelet derived growth factor AB
RCF: relative centrifugal force
RPM: revolutions per minute
VEGF: vascular endothelial growth factor
to concentrate platelets only, with little consideration for
the other constituents.2,3 Choukrouns protocol (Process
protocol, Nice, France) was simple and essentially consisted
of collecting venous blood into dry glass tubes, after which
the tubes would be spun at a low centrifuge speed to allow
the blood to separate into the constituents.4 This resulted
in three distinct layers forming in the blood collecting
tube, i.e. a red blood cell layer at the bottom of the tube,
an acellular layer at the uppermost part of the tube, and a
leukocyte- and platelet-rich fibrin (L-PRF) layer formed in
the middle of the tube.4 The L-PRF layer was considered as
the active biomaterial and has, since its development, been
promoted as an agent that accelerates wound healing and
tissue regeneration.5 Even though several clinical reports
have documented the use of L-PRF in oral and extra-oral
surgical procedures, controversy still exists with regards to
several aspects of this biomaterial. In this report we set out
to highlight some of these debates.
The terminology and classification of L-PRF
In an attempt to distinguish various platelet concentrates
from each other, Dohan Ehrenfest et al. used three key pa-
rameters i.e; the preparation process, the pharmacological
properties, and the characteristics of the final material to
establish a functional classification.6 By applying specific cri-
teria to these parameters, the authors were able to classify
platelet concentrates into four distinct categories (Table 1).6
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Table 1: Categories of platelet concentrates as proposed by
Dohan Ehrenfest et al,6
1. Pure platelet-rich plasma (P-PRP)
2. Leucocyte- and platelet-rich plasma (L-PRP)
3. Pure platelet-rich fibrin (P-PRF)
4. Leukocyte- and platelet-rich fibrin (L-PRF)
Although this classification elucidates and simplifies the
distinction of various platelet concentrates, it is not the
only existing proposed system to classify platelet concen-
trates.7 However, Dohan Ehrenfests classification is widely
quoted in the literature and at the time of its publication, in
2009, Choukrouns PRF was the only platelet concentrate
included in the category of L-PRF.
As the popularity of Choukrouns protocol for the produc-
tion of L-PRF grew, publications appeared describing proc-
esses that purported to produce L-PRF. However, none
had applied the exact criteria as described by Choukroun
(Process protocol, Nice, France).8-11 It is unclear whether
L-PRF produced by other than the Choukroun method
can be classified as a true L-PRF.4 Publications incor-
rectly use the terms L-PRF and Choukrouns PRF inter-
changeably, even though the exact method as described
by Choukroun has not been used to produce the platelet
concentrate.12 This has lead to incorrect assumptions and
a clear, unequivocal, classification of platelet concentrates
that is universally accepted is therefore sought. For the
purposes of clarity, the following proposed terminology
will be used throughout this article:
L-PRF Leukocyte- and platelet-rich fibrin. Defined as
a broad and all-inclusive category that is used to de-
scribe a mixed platelet, leukocyte and fibrin concen-
trate prepared using no-anticoagulants and a single
spin centrifuge technique.
L-PRF (C) Leukocyte- and platelet-rich fibrin (Choukroun
type). Defined as a specific leukocyte and platelet-rich fi-
brin prepared using Choukrouns protocol i.e. (the equip-
ment and the preparation method follows the exact rec-
ommended protocol as outlined by Choukroun1).
L- PRF (I/E) Leukocyte- and platelet-rich fibrin (In-
traspin/ EBA 20 type). Defined as a specific leukocyte
and platelet-rich fibrin prepared using either an Intra-
spin (Intra-Lock International, Boca-Raton, FL, USA), or
EBA 20 (Andreas Hettich GmbH & Co KG, Tuttlingen,
Germany) centrifuge and following the recommended
protocol as outlined by Dohan Ehrenfest et al.4
L-PRF (O) Leukocyte- and platelet-rich fibrin (Other).
Defined as a leukocyte- and platelet-rich fibrin prepared
in a manner similar to L-PRF (I/E) and L-PRF(C) produc-
tion, but using a non-purpose-built centrifuge.
Techniques and methods of
producing L-PRF
Choukrouns method of producing L-PRF was intended to
be a simple technique that would allow for the production
of high quality platelet and leukocyte concentrates, which
could be prepared easily and used in everyday healthcare
facilities.4 This method specified the use of a PC-02 table
centrifuge and a collection kit from Process (Nice, France).4
Further, the blood sample was to be taken without antico-
agulant in 10-mL blood collecting tubes which were then im-
mediately centrifuged at 3000 revolutions per minute (RPM)
(approximately 400g of relative centrifugal force (RCF)) for 10
minutes. The formed L-PRF clot could then be removed from
the blood collecting tube and used as required.
research <
299
The influence of centrifuge type and RCF on L-PRF
Even though Choukrouns protocol was clearly outlined,
a number of publications were subsequently produced
that reported on procedures which did not follow the pre-
scribed methods.13-15 Key to these differences was the fail-
ure to use a specific centrifuge (PC-O2, Process, Nice,
France) and a specific RCF (400g). In many publications,
the RCF was not reported, and instead the centrifuge
speed and time was quoted.13-15 This is a significant devia-
tion from the protocol since the influence of the RCF is
underestimated and not considered.
Relative centrifugal force (RCF) can be defined as the
amount of accelerative force applied to a sample in a cen-
trifuge.16 It is not equivalent to revolutions per minute (RPM)
and the terms cannot be used interchangeably. Centrifuges
work by putting samples in rotation around a fixed axis,
thereby applying an accelerative force perpendicular to the
axis.16 This resultant force causes the separation of vari-
ous elements in the sample based on the individual weight
of the sample elements and is the basis for blood separa-
tion techniques carried out by laboratory centrifuges. RCF
is measured in multiples of the standard acceleration due
to gravity at the Earths surface (x g) and is based on two
specific variables i.e. how wide the rotor is and how fast it
is moving.16 The radius of the centrifuge or rotor is as critical
to the process of producing a specific RCF as is the RPM.
Only those processes where the RPM and the rotor radius
are identical are comparable and any deviations from these
criteria may result in inaccuracies. Consequently, RCF will
only be constant for centrifuges with the same rotor radii.
Results derived from investigations using centrifuges with
different radii will produce differing RCFs.16 Therefore one
cannot assume that all centrifuges used for producing L-
PRF and spinning at 3000 RPM will produce an RCF of
400g. This is a significant parameter that is often misun-
derstood. RCF is critical to the production of L-PRF and
must be calculated for each centrifuge used, especially if
this parameter is not pre-set on the machine.
The effect of varying RCFs during platelet concentrate
preparation was recently reported.17 Amable, Carias, Teix-
eira et al. analysed various factors affecting the preparation
of Platelet-rich plasma (PRP), and showed that changes in
RCF significantly influenced the platelet yield even though
other parameters such as period of centrifuge time as well
as temperature remained constant. Dhurat and Sukesh
reviewed several PRP preparation methods.18 Based on
their analysis, it was shown that the use by authors of dif-
ferent RCF parameters resulted in variations in the platelet
yields of the PRP produced. More pertinently, scrutiny of
the literature reveals that although PRP has been clini-
cally used for several years, no standardised preparation
protocol has yet been documented. With regards to the
preparation methods of L-PRF that are published in the
literature, similar inconsistencies exist.
Other centrifuge parameters that may influence
L-PRF preparation.
A series of articles recently published by a team of authors
reported on investigations into the effect of various
parameters on the quality of the resultant L-PRFs.19-21
Using the same centrifugal force (400g) as well as the
same type of blood collecting tubes, the authors tested
four different commercially available L-PRF centrifuges.
The results indicated that centrifuge vibration as well as
centrifuge type significantly affect the quality and quantity
300 > research
of the L-PRF clot produced. Under scanning electron
microscope (SEM) analysis, the L-PRF clots produced
from the different centrifuges showed variations in cell
morphology and fibrin architecture, with some cells
showing signs of significant damage. These differences
were attributed to the type of centrifuge used.19-21 The
Intra-Spin L-PRF centrifuge (Intra-Lock International, Boca-
Raton, FL, USA) produced clots displaying cells with the
most stable and normal shape.20 It is therefore critical that
identical processes should be followed if the biomaterial
product is to be standardised. Researchers cannot simply
recreate the biomaterial by using any centrifuge with a
setting of 400g RCF even at the appropriate spin time.
Growth factors and their release kinetics
The preferred use of L-PRF in clinical practice is largely due
to its reported release of autogenous growth factors. It is
assumed that the high concentration of these growth fac-
tors results in reduced healing time as well as the stimu-
lation of tissue regeneration.8 These growth factors have
been well documented in the literature.22 Recently however,
the release kinetics of these growth factors has been ques-
tioned.23 Schr et al. prepared L-PRF (I/E) with a single spin
protocol at 400g for 12 minutes using an EBA 20 (Andreas
Hettich GmbH & Co KG, Tuttlingen, Germany) centrifuge.23
This is the same machine, recently upgraded, as the Intra-
Spin L-PRF centrifuge (Intra-Lock International, Boca-Raton,
FL, USA).24-26 The authors compared the release of various
growth factors from L-PRF, L-PRP and a coagulated blood
clot. The results demonstrated that the total growth factor
release of vascular endothelial growth factor (VEGF) as well
as of interleukin-1 (IL-1) was higher from the blood clot
than from any of the platelet concentrates. Furthermore, no
statistically significant differences could be established be-
tween the blood clot and the various platelet concentrates
as regards the amounts of insulin-like growth factor-1 (IGF-
1) and of platelet-derived growth factor AB (PDGF-AB) that
were released. The L-PRF (I/E) clot released the highest
concentrations of transforming growth factor 1 (TGF-1).
When the release kinetics of L-PRF (I/E), L-PRP and the
blood clot were investigated, the researchers found that the
various growth factors were released at different times as
well as for different lengths of time. An examination of this
effect on the migration on human bone marrow-derived
mesenchymal stem cells (MSC) and human umbilical vein
endothelial cells (HUVEC) found no significant differences
in the overall patterns of migration for any of the groups
tested. However it was reported that IGF-1 had a positive
correlation with the migration of both cell types whereas
PDGF-AB had a negative correlation with both cell types
i.e. MSC and HUVEC. It may be of relevance that IGF-1 had
the highest concentration in the blood clot and that there
were no differences in the release kinetics of this growth
factor when compared with those of L-PRF and L-PRP.23
In a similar study, in which the release of growth factors
as well as the effect of platelet concentrates on tendon
cells were compared with those of a whole blood clot, it
was shown that the platelet concentrates had the ability to
significantly increase cell proliferation as compared with
that of the blood clot.27 However, it must be pointed out
that the techniques of preparing these platelet concen-
trates were completely different between the two studies,
thereby influencing the final architecture and possible bio-
logical properties of the various concentrates.
Bone morphogenic proteins
Bone morphogenic proteins (BMPs) are low molecular
weight glycoproteins that are responsible for ectopic bone
formation.28 First described in the 1960s, these proteins
play a critical role in various aspects of cell function, dif-
ferentiation and tissue repair. More significantly, they are
crucial in the maintenance of skeletal integrity and bone
fracture healing. BMPs are released and synthesised by
a number of cells including osteoblasts, osteoprogenitor
cells, chondrocytes, platelets and macrophages.28,29 It is
therefore clear that the synthesis of these key proteins is
not restricted to bone forming cells.
It has recently been shown that L-PRF (I/E) releases BMP-2
over a period of seven days, but that the amounts released
are relatively small.21 Dohan Ehrenfest et al. found it difficult
to explain the exact origin of these BMPs, but suggested it
was related to the presence of leukocytes in the platelet con-
centrate.21 However it had been shown previously that the
platelets themselves contain significant amounts of BMP-2
and that the release of these proteins is pH dependent.30 As
a result, it has been suggested that the release of BMP-2
by platelets may play a significant role in the initial stages
of bone fracture healing, since the pH in this environment is
optimal for platelet activation.30
Other researchers have found that other BMPs such as
BMP-6, BMP-7 and BMP-4 are also released by platelets,
and, further, that the concentration of BMPs contained in
platelets is patient dependent.31,32 It has also been shown,
by genome-wide micro analysis, that lysed platelets have
the ability to upregulate proliferative pathways of osteob-
last like cells in-vitro.33
The potentially ground-breaking findings from various stud-
ies investigating the BMP potential of L-PRF as well as its
variants must be seen in the light of patient variation as well
as the pH of the test environment.21,28,30,32 Further research
into these factors may have clinical implications and could
explain the reasons for the inconsistent clinical outcomes
experienced when using platelet-rich concentrates for bone
grafting or regeneration. By implication then, it would cur-
rently be difficult to control the amounts of BMPs released
from platelets when used in a clinical setting.
Stem cells
Stem cells are undifferentiated cells that can differentiate
into specialized cells, including more stem cells or other
cell types during development.34 Recently, a variant of L-
PRF(C) has been analysed, thought to contain haemat-
opoetic stem cells (HSC).35 The presence of these HSC
cells is mostly determined using immunohistochemical
analysis for the detection of specific cell markers, in this
case, CD34. This is a transmembrane phosphoglycopro-
tein that is predominantly used as a marker for HSC as
well as haematopoetic progenitor cells.36 Although tradi-
tionally linked to cells of haematopoetic cell origin, CD34
has recently been linked to other non-haematopoetic cells
types such as mesenchymal stem cells (MSC), endothelial
progenitor cells and interstitial dendritic cells.36 Therefore,
the mere presence of CD34 positive cells does not allow
the assumption that a specific cell type such as HSC, ex-
ists. In order to verify the existence of HSC, the cells should,
in addition to the proven presence of CD34, display other
traits such as a low expression of CD90, a lack of expres-
sion of CD38 and human leukocyte antigen-DR (HLA-DR),
as well as a panel of mature lineage markers (lin-).36
www.sada.co.za / SADJ Vol 71 No. 7
The potential of CD34 positive cell types in L-PRF(C) and
its variants appears promising, but requires further inves-
tigation due to the variation in CD34 detection methods.
Almost all CD34 detection methods use antigen-antibody
interactions. These interactions are non-covalent and are
reversible, with the potential of affecting the detection of the
CD34 marker. Because of this, it is suggested that multiple
methods be used to verify the presence of CD34.36
Although peripheral blood has been used as a source for
CD34 positive cells in many forms of therapy, the baseline
concentration of these cells in peripheral blood is relatively
low.37,38 As such, most therapies that require the use of
CD34 positive cells enrich the presence of these cells in the
vasculature by using granulocyte colony-stimulating factor
(G-CSF).39 This allows for an adequate amount of cells to
be harvested for local or systemic transplantation. Whether
the levels of CD34 positive cells in L-PRF and its variants are
at therapeutic concentrations, requires further analysis.
Clinical results from studies using L-PRF
Several randomised controlled trials have been published
that involve the use of L-PRF in the clinical management
of a variety of disorders.40-43 These trials have contradic-
tory results, which may be related to a variation in the
techniques and equipment used to prepare L-PRF.41-45
Nevertheless, several articles report positive clinical out-
comes even when standardised preparation techniques
have not been used.46-49 Whilst most of these publications
are in fact case reports, nevertheless even randomised
controlled trials where the RCF has not been verified,
have shown positive clinical outcomes.43 This is similar to
reports about the use of PRP, which show a variety of
clinical results based on the various methods of producing
PRP.18 Further research is therefore required to verify clini-
cal differences which may be associated with the various
methods of preparing L-PRF.
Does generic L-PRF exist?
L-PRF (O) is prepared using standard blood collecting
tubes and a non-purpose built table top centrifuge deliver-
ing an RCF of 400g with a centrifuge time of 12 minutes.
This method allows for the preparation of a platelet and
leukocyte concentrate without the need for specialised
equipment. Several case reports have demonstrated posi-
tive clinical results when using this preparation method.50,51
However one cannot assume that this generic type of L-PRF
has properties similar to those of L-PRF(C) or to L-PRF(I/E)
since it has previously been shown that the centrifuge type
may play a significant role in the final morphological fea-
tures of the end product.19,20 Further analysis of this bioma-
terial is therefore required to determine equivalence to the
established protocols for L-PRF preparation.
Conclusions
The concept of L-PRF as a bioactive material with possi-
ble regenerative properties has resulted in it being adopted
for use in various clinical procedures. However, the wide-
spread use of this material has resulted in the production
of several variants. Whether the biological properties of all
these variants are similar, is unknown. Contradictory clini-
cal results are reported in the literature with several generic
types of L-PRF showing diverse results. In order to minimise
the controversies associated with L-PRF, further research
is required to determine which factors affect the biological
properties of the material and whether these factors are
clinically beneficial and relevant.
research <
301
Disclosure policy
The authors declare no conflict of interest regarding the publica-
tion of this paper. This paper forms part of the requirements for
partial fulfilment of the specifications for the degree PhD.
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